Aptamer-based label-free detection of PDGF using ruthenium(II) complex as luminescent probe.

We report a simple, cost-effective, and label-free detection method, consisting of a platelet-derived growth factor (PDGF) binding aptamer and hydrophobic Ru(II) complex as a sensor system for PDGF. The binding of PDGF with the aptamer results in the weakening of the aptamer-Ru(II) complex, monitored by luminescence signal. A substantial enhancement in the luminescence intensity of Ru(II) complex is observed in the presence of aptamer due to the hydrophobic interaction. Upon addition of PDGF, the luminescence intensity is decreased, due to the stronger interaction between the aptamer and PDGF resulting in the displacement of Ru(II) complex to the aqueous solution. Our assay can detect a target specifically in a complex medium such as the mixture of proteins, at a concentration of 0.8 pM.

Sphenoid mucocele: an uncommon complication of a rare condition.

A 58-year-old white woman presented with sudden onset of diplopia, headache and vomiting with a history of tiredness and lethargy over the past four to six months. She had smooth, pale, hairless skin and on examination she was found to have left-sided third and sixth nerve palsy. Laboratory tests confirmed pan-anterior hypopituitarism. Computerized tomography scan of head and magnetic resonance imaging appearances were consistent with those of a sphenoid sinus mucocoele. Following adequate replacement with hydrocortisone and thyroxine she underwent sphenoid mucocoele drainage and endoscopic left sphenoethmoidectomy. Her symptoms were relieved over the next few days and she had a near-total recovery of ophthalmoplegia over the following three months. Pituitary function tests showed partial resolution of hypopituitarism with recovery of hypothalamic-pituitary-adrenal axis and hydrocortisone therapy was withdrawn, but she continued to require thyroxine.

The Diatom EST Database.

The Diatom EST database provides integrated access to expressed sequence tag (EST) data from two eukaryotic microalgae of the class Bacillariophyceae, Phaeodactylum tricornutum and Thalassiosira pseudonana. The database currently contains sequences of close to 30,000 ESTs organized into PtDB, the P.tricornutum EST database, and TpDB, the T.pseudonana EST database. The EST sequences were clustered and assembled into a non-redundant set for each organism, and these non-redundant sequences were then subjected to automated annotation using similarity searches against protein and domain databases. EST sequences, clusters of contiguous sequences, their annotation and analysis with reference to the publicly available databases, and a codon usage table derived from a subset of sequences from PtDB and TpDB can all be accessed in the Diatom EST Database. The underlying RDBMS enables queries over the raw and annotated EST data and retrieval of information through a user-friendly web interface, with options to perform keyword and BLAST searches. The EST data can also be retrieved based on Pfam domains, Cluster of Orthologous Groups (COG) and Gene Ontologies (GO) assigned to them by similarity searches. The Database is available at http://avesthagen.sznbowler.com.

Practical applications of Beam's Eye View-based treatment planning to head and neck sites.

Because of the complex anatomy of the structures involved by tumor as well as of the critical normal structures, treatment planning for advanced or spatially irregular cancers of head and neck sites is often extremely challenging. Computerized axial tomographic scanning is frequently invaluable in delineating tumor extension into areas inaccessible to physical examination. Our Beam's Eye View Planning (BEVP) capability allows us to incorporate this radiographic information accurately into actual plans used in the clinic. Over the past 2 1/2 years, we have applied this technique to 31 selected head and neck cancer patients at Michael Reese/University of Chicago Center for Radiation Therapy. Tumors were chosen on the basis of anatomical complexity: most involved multiple head and neck sites including orbit, skull base, paranasal sinuses and cavernous sinus. In all cases, radiation tolerance of critical normal structures including spinal cord, brain stem, optic chiasm, and eye had to be considered. With careful use of rigid immobilization devices and the outlining of several normal structures for purposes of alignment, we had no difficulty applying our BEVP technique to clinical simulations. Oblique field blocking was especially facilitated by BEVP. We found the BEVP technique very useful to assure that tumor coverage was adequate and tolerance of normal tissues not exceeded.

Online repositioning during treatment of the prostate: a study of potential limits and gains.

PURPOSE: With on-line portal imaging devices and image registration tools, the verification of radiation field position prior to each treatment becomes technically feasible. In this paper, we analyze the impact of pre-treatment verification and field position adjustment on target coverage and normal tissue sparing. METHODS AND MATERIALS: Port films were compared with corresponding simulation films to determine the magnitude of setup variations in patients treated for prostate cancer. From these data, an analytic function was determined between geometric coverage of the target and field margin size. A paradigm for on-line patient repositioning was employed to generate a new relationship between margin and target coverage. Margins were selected for the situations of normal treatment and on-line repositioning to ensure target coverage. Dose-volume histograms were generated for a typical prostate treatment using these margins. RESULTS: On-line repositioning, when setup errors exceed 1 cm, results in a 6 mm reduction in margin, suggesting that 10% of the volume of bladder and rectum may be spared of high dose. CONCLUSION: The use of on-line imaging and image registration to guide adjustment of patient setup may lead to a reduction in the volume of normal tissues irradiated, and possibly improve the probability of complication-free survival in future treatments.

"Instant-mix" whole brain photon with neutron boost radiotherapy for malignant gliomas.

From July 1985 through March 1987, 44 consecutive patients with supratentorial, nonmetastatic anaplastic astrocytoma (AA) and glioblastoma multiforme (GBM) were treated with whole brain photon irradiation with concomitant neutron boost at the University of Chicago. All patients had biopsy proven disease and surgery ranged from biopsy to total gross excision. Whole brain photon radiation was given at 1.5 Gy per fraction, 5 days weekly for a total dose of 45 Gy in 6 weeks. Neutron boost radiation was prescribed to a target minimum dose that included the pre-surgical CT tumor volume plus 1 cm margin. Neutrons were administered 5-20 minutes prior to photon radiation twice weekly and a total dose of 5.2 Gyn gamma was administered over 6 weeks. Median follow-up was 36 months. The median survival was 40.3 months for anaplastic astrocytoma (10 patients) and 11 months for glioblastoma multiforme (34 patients) and 12 months for the overall group. Variables that predicted longer median survival included histology (AA vs. GBM), age (less than or equal to 39 years vs. older), and extent of surgery (total gross or partial excision vs. biopsy) whereas tumor size and Karnofsky performance status did not have a significant influence. The median survival of the anaplastic astrocytoma group was better than expected compared to the RTOG 80-07 study (a dose-finding study of similar design to this study) and historical data. Reasons for this are discussed.

Quantitative imaging study of extent of surgical resection and prognosis of malignant astrocytomas.

OBJECTIVE: This study used quantitative radiological imaging to determine the effect of surgical resection on postoperative survival of patients with malignant astrocytomas. Previous studies relied on the surgeons' impressions of the amount of tumor removed, which is a less reliable measure of the extent of resection. METHODS: Information concerning possible prognostic factors was collected for 75 patients undergoing magnetic resonance imaging or computed tomography preoperatively and within 10 days postoperatively. Image analysis of the neuroradiological studies was conducted to quantify pre- and postoperative total tumor volumes and enhancing volumes. Univariate and multivariate proportional hazards models were used to analyze the regression of survival regarding 22 covariates that might affect survival. The covariates that were entered included age, gender, tumor grade, cumulative radiation dose, chemotherapy, seizures as a first symptom, Karnofsky performance status at presentation, pre- and postoperative total and enhancing tumor volumes, ratio of pre- to postoperative total and enhancing tumor volumes, tumor location, surgeon's impression of the degree of resection, and subsequent surgery. RESULTS: There were 23 patients with anaplastic astrocytomas and 52 with glioblastomas multiforme. The estimated mean survival time was 27 months for patients undergoing gross total resection, 33 months for subtotal resection, and 13 months for open or stereotactic biopsy. Five factors that were significant predictors of survival in multivariate analysis were tumor grade, age, Karnofsky performance status, radiation dose, and postoperative complications (P < 0.05). In univariate analysis, tumor grade, radiation dose, age, Karnofsky status, complications, presence of enhancing tumor in postoperative imaging, and postoperative volume of enhancing tumor were significantly associated with survival (P < 0.05). CONCLUSION: We conclude that the most important prognostic factors affecting survival of patients with anaplastic astrocytomas and glioblastomas multiforme are tumor grade, age, preoperative performance status, and radiation therapy. Postoperative complications adversely affect survival. Aggressive surgical resection did not impart a significant increase in survival time. Surgical resection may improve survival, but its importance is less than that of other factors and may be demonstrable only by larger studies.

Acute corneal endothelial changes after laser in situ keratomileusis.

PURPOSE: To report the acute effects of laser in situ keratomileusis (LASIK) on the corneal endothelium. METHODS: Twenty eyes of 10 consecutive patients (mean age, 38.1 +/- 10.84 years) underwent bilateral simultaneous LASIK for myopic astigmatism (spherical equivalent ranging from -1.75 to -7.13 diopters) without any complications. Each eye was evaluated by slit-lamp biomicroscopy and noncontact specular microscopy preoperatively, within 15 minutes after LASIK and 1 day after surgery. Specular microscopy images were then analyzed to calculate endothelial cell density (ECD), coefficient of variation (CV) of cell size, and percentage of hexagonal cells. RESULTS: All corneas demonstrated marked alterations in endothelial cell morphology by slit-lamp biomicroscopy within 15 minutes after surgery that resolved by the first postoperative day. Central corneal endothelial analysis by noncontact specular microscopy confirmed pleomorphism with definite loss of hexagonality. Mean ECD was calculated to be 2,816.3 +/- 286.02 cells/mm(2) preoperatively, 2,750.85 +/- 327.95 cells/mm(2) on day 0 (p = 0.395), and 2,810.55 +/- 218.48 cells/mm(2) on day 1 (p = 0.461). Mean CV was 32.65 +/- 7.29 preoperatively, 34.4 +/- 6.19 on day 0 (p = 0.412), and 30.9 +/- 5.54 on day 1 (p = 0.067). Mean percentage of hexagonal cells was 63.35 +/- 10.76 preoperatively, 47.55 +/- 9.69 on day 0 (p = 0.000009), and 60 +/- 9.3 on day 1 (p = 0.00003). CONCLUSION: Qualitative and quantitative changes in endothelial cell morphology (i.e., decreased endothelial cell hexagonality) demonstrate that LASIK does induce an acute effect on the corneal endothelium that may represent transient endothelial cell edema.

Quantitation of treatment volumes from CT and MRI in high-grade gliomas: implications for radiotherapy.

Long-term survival of patients with high-grade gliomas remains extremely poor. The main reason for such an outcome is local failure, or recurrence, after surgery and/or radiotherapy. Higher doses of radiation may result in decreased local failure rates provided that the location (and extent) of gross tumor and microscopic disease can be defined accurately. The abnormalities appearing in images from diagnostic modalities, such as CT and MRI, are being used as a starting point and as a guide for the clinical definition of tumor and its extensions. However, some recent studies on two-dimensional specimens, correlating histopathological findings to CT and MRI images, showed that the resulting definition of tumor cell extensions was unsatisfactory, different, and in need of ample margins. We carried out a retrospective analysis to compare the target volumes that would have been defined by CT, T2-weighted MRI, and T1-weighted postgadolinium MRI images of the same individual and to explore the implications of the resulting volume definitions for radiotherapy. The results of our limited study, based on the margins used, indicate that the CT-defined target volume is consistently larger than that from either of the two MRI modalities and suggest that noncoplanar approaches for its treatment and other local approaches for tumor boost should be considered. We conclude that until more definitive histopathological guidelines correlated to image features have been formulated and agreed upon, one should try to make full use of all available diagnostic information in order to minimize the possibility of geographical miss of target extensions.

The impact on survival by adjuvant chemotherapy and radiation therapy in stage II non-small-cell lung cancer.

Forty-nine consecutive patients with pathologic Stage II non-small-cell lung cancer treated over a 15-year period were retrospectively reviewed. The treatment strategy evolved during the period of review. Early patients were treated with surgery alone (S); subsequent patients were treated with adjuvant radiation therapy (SR); and more recent patients were treated with postoperative chemotherapy and radiation therapy (SRC). Fifteen patients received S alone, 10 patients received SR, and 24 patients received SRC. The median survival time (MST) of all 49 patients was 20 months, and the estimated 5-year survival was 25%. The MST of patients in each of the three treatment arms was S-6 months; SR-19 months; and SRC-25 months. The majority of patients died from systemic relapses or second primary lung cancers. The addition of adjuvant therapy (SR, SRC) significantly improved the MST of patients compared to surgery alone (S). The overall survival of patients did not change between treatment arms.

Characterization of the radish mitochondrial orfB locus: possible relationship with male sterility in Ogura radish.

The orfB locus of the normal (fertile) and Ogura (male-sterile) radish mitochondrial genomes has been characterized in order to determine if this region, which has previously been correlated with cytoplasmic male sterility (CMS) in Brassica napus cybrids (Bonhomme et al. 1991; Temple et al. 1992), could also be involved in radish CMS. In normal radish, orfB is expressed as a 600-nucleotide (nt) transcript. In Ogura radish, orfB is present as the second gene of a 1200-nt transcript that also contains a 138-codon open reading frame (orf138). Sequences showing similarity to orf138 are present in normal radish, but are not expressed.

Organ-specific reduction in the abundance of a mitochondrial protein accompanies fertility restoration in cytoplasmic male-sterile radish.

The mitochondrial DNA of plants containing the male sterility-causing Ogura cytoplasm of radish contain a novel gene, orf138, that is transcribed as part of a bicistronic mRNA. Genetic studies have previously linked male sterility with the orf138 locus. To determine if orf138 is expressed at the protein level, and investigate the effect of fertility restoration on ORF138 levels, we have raised antibodies to an ORF138-glutathione S-transferase fusion protein. Anti-ORF138 antibodies detect a 20 kDa protein that is associated with the mitochondrial membrane of sterile Ogura radish plants. Nuclear restoration is accompanied by a dramatic reduction in the amount of this protein in mitochondria of flowers and leaves, but not roots of fertile Ogura radish plants. The presence or absence of fertility restoration genes has no detectable effect on the size, abundance, or RNA editing patterns of orf138 transcripts. These results support genetic studies that have implicated orf138 in Ogura cytoplasmic male sterility and suggest that the restorer genes may be affecting either the translation or stability of ORF138.

Heat shock response of the chloroplast genome in Vigna sinensis.

The light-dependent protein synthesis occurring in chloroplasts isolated from the leaves of 7-day-old Vigna sinensis responded to heat shock with the induction of a set of four heat shock proteins (HSPs). The synthesis of these four HSPs at the elevated temperature was transcriptionally regulated, and all of them were found to be thylakoid membrane-bound. The synthesis of these chloroplast-coded HSPs was also observed in etioplasts during protein synthesis at the elevated temperatures with the exogenously added ATP. The in vivo induction of these plastid-coded HSPs was observed only in the leaves subjected to gradual increase in temperature but not in leaves subjected to rapid heat shock. Further, the in vitro synthesis of these plastid-coded HSPs was seen only in chloroplasts isolated from the control or from leaves subjected to gradual increase in temperature and not in chloroplasts isolated from leaves subjected to rapid heat shock. Taken together these observations suggest that in V. sinensis, the plastid genome contains a definite heat shock response and the in vivo expression of this response corresponds to the gradual rise in temperature normally occurring under field conditions.

Subunit 6 of the Fo-ATP synthase complex from cytoplasmic male-sterile radish: RNA editing and NH2-terminal protein sequencing.

RNA editing and NH2-terminal processing of subunit 6 (atp6) of the mitochondrial Fo-ATPase complex has been investigated for the normal (fertile) and Ogura (male-sterile) radish cytoplasms to determine if previously identified differences between the Ogura atp6 locus and its normal radish counterpart are associated with cytoplasmic male sterility. Analysis of cDNA clones from five different sterile and fertile radish lines identified one C-to-U transition, which results in the replacement of a proline with a serine, in several of the lines. No editing of atp6 transcripts was observed in two lines, Scarlet Knight (normal radish) and sterile CrGC15 (Ogura radish). This is the first example of a naturally occurring plant mitochondrial gene that is not edited. The Ogura atp6 polypeptide is synthesized with a predicted NH2-terminal extension of 174 amino acids in contrast to the nine amino acid extension found in normal radish. In spite of the lack of similarity between the two extensions, NH2-terminal sequence analysis indicates that both polypeptides are processed to yield identical core proteins with a serine as the NH2-terminal residue. These results indicate that ATPase subunit 6 is synthesized normally in Ogura radish, and that it is unlikely that the atp6 locus is associated with Ogura cytoplasmic male sterility.

Induced corneal astigmatism after macular translocation surgery with scleral infolding.

OBJECTIVE: To document the corneal astigmatism that occurs with macular translocation after scleral infolding surgery. DESIGN: Retrospective case series of a nonrandomized clinical trial. PARTICIPANTS: Eight consecutive age-related macular degeneration patients (eight eyes) with choroidal neovascularization who underwent macular translocation with scleral infolding at the Duke University Eye Center from December 1998 through October 1999. METHODS: We retrospectively reviewed the charts of eight consecutive patients who underwent macular translocation surgery involving scleral infolding in the superotemporal quadrant. Two patients subsequently underwent release of scleral infolding. MAIN OUTCOME MEASURES: After surgery, these eyes were evaluated for corneal astigmatism with manifest refraction, keratometry, and computerized corneal topography. RESULTS: All eight eyes of eight patients revealed marked degrees of corneal astigmatism. Measurement of astigmatism via manifest refraction, keratometry, and corneal topography confirmed postoperative astigmatism corresponding to the axis of the scleral infolding. The amount of corneal astigmatism ranged from 1.75 to 7.37 diopters (D; mean, 4.60 D), with steepening along the axis of scleral infolding in the superotemporal quadrant of each eye (mean, 42.50 degrees from vertical; range, 24 degrees -66 degrees from vertical). Release of scleral infolding in two patients resulted in significant reduction of corneal astigmatism. CONCLUSIONS: Scleral shortening procedures used in macular translocation surgery may induce large amounts of corneal astigmatism. These patients should be assessed with keratometry and corneal topography to determine the accurate amount and axis. Thereafter, contact lens fitting or scleral infolding release may be considered as therapeutic options for large amounts of astigmatism persisting after surgery.

Separation of alveolar surfactant into subtypes. A comparison of methods.

Alveolar surfactant is known to exist in several morphologic forms or subtypes which have been separated from bronchoalveolar lavage fluid (BAL) by two types of methods-differential centrifugation (DC) and equilibrium buoyant density gradient centrifugation (EBDC). DC separates BAL into large aggregates (LA) and small aggregates (SA); EBDC separates BAL into three peaks called ultraheavy (UH), heavy (H), and light (L). We compared these two separation methods by subjecting replicates of the same pools of BALF from groups of mice to DC and EBDC in parallel assays. We found that each method was highly internally consistent, but that the amount of phospholipid in the LA fraction of DC was consistently and substantially less (by 33 to 43%) than that found in the UH + H fractions of EBDC. This appeared to be due to failure of DC to sediment all of the phospholipid that banded as UH or H in EBDC despite adjustments in the time and g-force of DC. In experiments where differentially labeled purified H and L subtypes were subjected to DC over a wide range of g-force and time conditions, cross-contamination of the DC pellet and supernatant with heterologous subtypes was always present (4 to 33% cross-contamination). Addition of extraneous serum proteins to the BAL, as a model of lung damage, resulted in further inconsistencies in DC but not EBDC. Investigators may wish to bear these considerations in mind when planning or interpreting the results of experiments bearing on surfactant subtype analysis.

Protein-lipid interactions and enzyme requirements for light subtype generation on cycling reconstituted surfactant.

Surfactant convertase is required for conversion of heavy density (H) natural surfactant to light density (L) subtype during cycling in vitro, a technique that reproduces surfactant metabolism. To study mechanisms of H to L conversion, we prepared liposomes of dipalmitoylphosphatidylcholine (DPPC) and phosphatidylglycerol (PG), or the phospholipids (PL) in combination with either surfactant protein A (SP-A), surfactant protein B (SP-B), or both SP-A and SP-B. Phospholipids alone showed time-dependent conversion from heavy to light subtype on cycling in the absence of convertase, which was decreased by adding SP-B, but not SP-A, to phospholipids (p < 0.01 for PL+SP-B, or PL+SP-A+SP-B vs. PL, or PL+SP-A). The ultrastructure, surface activity, buoyant density, and L subtype generation on cycling PL+SP-A+SP-B with partially purified convertase or with phospholipase D were similar to those of natural TM. In conclusion, a reconstituted surfactant mimics the behavior of natural surfactant on cycling, and reveals that interaction of SP-B with phospholipids decreases L subtype generation. In addition, esterase/ phospholipase D activity is required for conversion of heavy to light subtype on cycling.

Molecular characterization of glyoxalase II from Arabidopsis thaliana.

Glyoxalase II is part of the glutathione-dependent glyoxalase detoxification system. In addition to its role in the detoxification of cytotoxic 2-oxo-aldehydes, specifically methylglyoxal, it has been suggested that the glyoxalase system may also play a role in controlling cell differentiation and proliferation. During the analysis of a T-DNA-tagged mutant of Arabidopsis we identified the gene for a glyoxalase II isozyme (GLY1) that appears to be mitochondrially localized. The cDNA encoding a glyoxalase II cytoplasmic isozyme (GLY2) was also isolated and characterized. Southern blot and sequence analyses indicate that glyoxalase II proteins are encoded by at least two multigene families in Arabidopsis. Escherichia coli cells expressing either GLY1 or GLY2 exhibit increased glyoxalase II activity, confirming that they do, in fact, encode glyoxalase II proteins. Northern analysis shows that the two genes are differentially expressed. Transcripts for the mitochondrial isozyme are most abundant in roots, while those for the cytoplasmic isozyme are highest in flower buds. The identification of glyoxalase II isozymes that are differentially expressed suggests that they may play different roles in the cell.

Influence of phospholipid composition on the properties of reconstituted surfactants.

The influence of phospholipids on the ultrastructure and metabolism of reconstituted surfactants has not been well defined. The aim of this study was to determine if changes in the phospholipid composition of reconstituted surfactants altered their biophysical properties, ultrastructure, and conversion to light subtype by cycling. We prepared various surfactants containing radiolabeled dipalmitoylphosphatidylcholine ([14C]DPPC). The addition of phosphatidylglycerol (PG) or dipalmitoylphosphatidic acid (PA) to DPPC increased conversion to light subtype. In contrast, the addition of dipalmitoylphosphatidylglycerol (DPPG) to DPPC markedly reduced conversion to light subtype on cycling. DPPC and DPPC+PG produced large liposomes ( approximately 1,000 nm), whereas DPPC+PA or DPPC+DPPG formed multilamellar membranes. Mixtures of DPPC and PA were highly surface active in vitro, whereas the surface activity of DPPC+DPPG was similar to that of DPPC. In conclusion, the ultrastructure, metabolism, and surface active properties of DPPC+PG mixtures were influenced markedly by alterations in the fatty acid composition or polar head group of PG.

Lung "surfactant convertase" is a member of the carboxylesterase family.

The extracellular conversion of lung surfactant from tubular myelin to the small vesicular form has previously been shown to require a serine-active enzyme called "surfactant convertase." In the present study, a 72kD serine-active enzyme previously identified in mouse lung alveolar lavage and having convertase activity was partially sequenced. Sixty-eight residues obtained from amino acid sequencing of this protein show that it is a new member of the mouse carboxylesterase family (EC 3.1.1.1). The 72kD lung protein also has esterase activity. A commercial esterase of the same family was able to reproduce surfactant convertase bioactivity in vitro, unlike several serine proteinases previously tested. We conclude that surfactant convertase is a carboxylesterase which mediates a biochemical step in the extracellular metabolism of surfactant.